Lettuce variety cardinal

ABSTRACT

The present invention provides novel lettuce cultivar Cardinal and plant parts, seed, and tissue culture therefrom. The invention also provides methods for producing a lettuce plant by crossing the lettuce plants of the invention with themselves or another lettuce plant. The invention also provides lettuce plants produced from such a crossing as well as plant parts, seed, and tissue culture therefrom.

FIELD OF THE INVENTION

This invention is in the field of lettuce plants.

BACKGROUND OF THE INVENTION

The present invention relates to a lettuce (Lactuca sativa L.) varietydesignated Cardinal.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. Lactuca sativa is in the Cichoreae tribe of theAsteraceae (Compositae) family. Lettuce is related to chicory,sunflower, aster, dandelion, artichoke, and chrysanthemum. Sativa is oneof about 300 species in the genus Lactuca. There are seven differentmorphological types of lettuce. The crisphead group includes the icebergand batavian types. Iceberg lettuce has a large, firm head with a crisptexture and a white or creamy yellow interior. The batavian lettucepredates the iceberg type and has a smaller and less firm head. Thebutterhead group has a small, soft head with an almost oily texture. Theromaine, also known as cos lettuce, has elongated upright leaves forminga loose, loaf-shaped head and the outer leaves are usually dark green.Leaf lettuce comes in many varieties, none of which form a head, andinclude the green oak leaf variety. Latin lettuce looks like a crossbetween romaine and butterhead. Stem lettuce has long, narrow leaves andthick, edible stems. Oilseed lettuce is a type grown for its large seedsthat are pressed to obtain oil. Latin lettuce, stem lettuce, and oilseedlettuce are seldom seen in the United States.

Presently, there are over one thousand known lettuce cultivars. As acrop, lettuce is grown commercially wherever environmental conditionspermit the production of an economically viable yield.

Lettuce, in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, there is an ongoing need for improvedlettuce varieties.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel lettuce cultivardesignated Cardinal, also known as LRLU20-2023, having desirablecharacteristics including short core length, bolting tolerance, andresistances to Bremia lactucae strains BI: BL: 5-9US, Tomato Bushy Stuntvirus, and Nasonovia ribisnigri biotype Nr: 0. The invention alsoencompasses the seeds of lettuce cultivar Cardinal, the plants oflettuce cultivar Cardinal, plant parts of the lettuce cultivar Cardinal(including leaves, seed, gametes), methods of producing seed fromlettuce cultivar Cardinal, and method for producing a lettuce plant bycrossing the lettuce cultivar Cardinal with itself or another lettuceplant, methods for producing a lettuce plant containing in its geneticmaterial one or more transgenes, and the transgenic lettuce plantsproduced by that method. The invention also relates to methods forproducing other lettuce plants derived from lettuce cultivar Cardinaland to lettuce plants, parts thereof and seed derived by the use ofthose methods. The present invention further relates to hybrid lettuceseeds and plants (and parts thereof including leaves) produced bycrossing lettuce cultivar Cardinal with another lettuce plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Cardinal. In embodiments, thetissue culture is capable of regenerating plants having all oressentially all of the physiological and morphological characteristicsof the foregoing lettuce plant and/or of regenerating plants having thesame or substantially the same genotype as the foregoing lettuce plant.In exemplary embodiments, the regenerable cells in such tissue culturesare meristematic cells, cotyledons, hypocotyl, leaves, pollen, embryos,roots, root tips, anthers, pistils, ovules, shoots, stems, petiole,pith, flowers, capsules and/or seeds as well as callus and/orprotoplasts derived from any of the foregoing. Still further, thepresent invention provides lettuce plants regenerated from the tissuecultures of the invention.

As a further aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a plant of lettuce cultivarCardinal with itself or a second lettuce plant. Optionally, the methodfurther comprises collecting the seed.

Another aspect of the invention provides methods for producing hybridsand other lettuce plants derived from lettuce cultivar Cardinal. Lettuceplants derived by the use of those methods are also part of theinvention as well as plant parts, seed, gametes and tissue culture fromsuch hybrid or derived lettuce plants.

In representative embodiments, a lettuce plant derived from lettucecultivar Cardinal comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Cardinal. In embodiments, alettuce plant or population of lettuce plants derived from lettucecultivar Cardinal comprises, on average, at least 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of its alleles (i.e., theoretical allelic content; TAC)from lettuce cultivar Cardinal, e.g., at least about 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of the genetic complement of lettuce cultivar Cardinal,and optionally is the result of a breeding process comprising one or twobreeding crosses and one or more of selfing, sibbing, backcrossingand/or double haploid techniques in any combination and any order. Inembodiments, the breeding process does not include a breeding cross, andcomprises selfing, sibbing, backcrossing and or double haploidtechnology. In embodiments, the lettuce plant derived from lettucecultivar Cardinal is one, two, three, four, five or more breedingcrosses removed from lettuce cultivar Cardinal.

In embodiments, a hybrid or derived plant from lettuce cultivar Cardinalcomprises a desired added trait(s). In representative embodiments, alettuce plant derived from lettuce cultivar Cardinal comprises at least10 of the morphological and physiological characteristics of lettucecultivar Cardinal (e.g., as described in Table 1 and Tables 2-12). Inembodiments, the lettuce plant derived from lettuce cultivar Cardinalcomprises essentially all of the morphological and physiologicalcharacteristics of lettuce cultivar Cardinal (e.g., cultivar Cardinalcomprises at least 10 of the morphological and physiologicalcharacteristics of lettuce cultivar Cardinal (e.g., as described inTable 1 and Tables 2-12).

The invention also relates to methods for producing a lettuce plantcomprising in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods (and progeny lettuceplants comprising the transgene). Also provided are plant parts, seedand tissue culture from such transgenic lettuce plants, optionallywherein one or more cells in the plant part, seed, or tissue culturecomprises the transgene. The transgene can be introduced via planttransformation and/or breeding techniques.

In another aspect, the present invention provides for single locusconverted plants of lettuce cultivar Cardinal. Plant parts, seed, andtissue culture from such single locus converted plants are alsocontemplated by the present invention. The single locus may be adominant or recessive allele. In representative embodiments, the singletransferred locus confers such traits as male sterility, herbicideresistance, pest resistance (e.g., insect and/or nematode resistance),modified fatty acid metabolism, modified carbohydrate metabolism,disease resistance (e.g., for bacterial, fungal and/or viral disease),male fertility, enhanced nutritional quality, improved appearance (e.g.,color), improved salt tolerance, industrial usage, or any combinationthereof. The single locus may be a naturally occurring lettuce locus, agenome edited locus, a mutated locus (e.g., chemically or radiationinduced), or a transgene introduced into lettuce through geneticengineering techniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding, for example, recurrent selection, backcrossing, pedigreebreeding, double haploid techniques, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selectionand/or transformation. Seeds, lettuce plants, and parts thereof,produced by such breeding methods are also part of the invention.

The invention also provides methods of multiplication or propagation oflettuce plants of the invention, which can be accomplished using anymethod known in the art, for example, via vegetative propagation and/orseed.

The invention further provides a method of producing food or feedcomprising (a) obtaining a lettuce plant of the invention, optionallywherein the plant has been cultivated to maturity, and (b) collecting atleast one lettuce plant or part thereof (e.g., leaves) from the plant.

Additional aspects of the invention include harvested products andprocessed products from the lettuce plants of the invention. A harvestedproduct can be a whole plant or any plant part, as described herein.Thus, in some embodiments, a non-limiting example of a harvested productincludes a seed, a leaf and/or a stem.

In representative embodiments, a processed product includes, but is notlimited to: cut, sliced, ground, pureed, dried, canned, jarred, washed,packaged, frozen and/or heated leaves and/or seeds of the lettuce plantsof the invention, or any other part thereof. In embodiments, theprocessed product includes washed and packaged leaves (or parts thereof)of the invention.

The seed of the invention can optionally be provided as an essentiallyhomogenous population of seed of a single plant or cultivar. Essentiallyhomogenous populations of seed are generally free from substantialnumbers of other seed, e.g., at least about 90%, 95%, 96%, 97%, 98% or99% pure.

In representative embodiments, the invention provides a seed of lettucecultivar Cardinal.

As a further aspect, the invention provides a plant of lettuce cultivarCardinal.

As an additional aspect, the invention provides a lettuce plant, or apart thereof, having all or essentially all of the physiological andmorphological characteristics of a plant of lettuce cultivar Cardinal.

As another aspect, the invention provides leaves and/or seed of thelettuce plants of the invention and a processed product from the leavesand/or seed of the inventive lettuce plants.

As still another aspect, the invention provides a method of producinglettuce seed, the method comprising crossing a lettuce plant of theinvention with itself or a second lettuce plant. The invention alsoprovides seed produced by this method and plants produced by growing theseed.

As yet a further aspect, the invention provides a method for producing aseed of a lettuce plant derived from lettuce cultivar Cardinal, themethod comprising: (a) crossing a lettuce plant of lettuce cultivarCardinal with a second lettuce plant; and (b) allowing seed of a lettuceplant derived from lettuce cultivar Cardinal to form. In embodiments,the method further comprises: (c) growing a plant from the seed derivedfrom lettuce cultivar Cardinal of step (b); (d) selfing the plant grownfrom the lettuce seed derived from lettuce cultivar Cardinal or crossingit to a second lettuce plant to form additional lettuce seed derivedfrom lettuce cultivar Cardinal, and (e) repeating steps (c) and (d) 0 ormore times to generate further derived lettuce seed. Optionally, themethod comprises: (e) repeating steps (c) and (d) one or more times(e.g., one to three, one to five, one to six, one to seven, one to ten,three to five, three to six, three to seven, three to eight or three toten times) to generate further derived lettuce plants. As anotheroption, the method can comprise collecting the seed. The invention alsoprovides seed produced by these methods and plants produced by growingthe seed.

As another aspect, the invention provides a method of producing lettuceleaves, the method comprising: (a) obtaining a plant of lettuce cultivarCardinal, optionally wherein the plant has been cultivated to maturity;and (b) collecting leaves from the plant. The invention also providesthe leaves produced by this method.

Still further, as another aspect, the invention provides a method ofvegetatively propagating a plant of lettuce cultivar Cardinal. In anon-limiting example, the method comprises: (a) collecting tissuecapable of being propagated from a plant of lettuce cultivar Cardinal;(b) cultivating the tissue to obtain proliferated shoots; and (c)rooting the proliferated shoots to obtain rooted plantlets. Optionally,the invention further comprises growing plants from the rootedplantlets. The invention also encompasses the plantlets and plantsproduced by these methods.

As an additional aspect, the invention provides a method of introducinga desired added trait into lettuce cultivar Cardinal, the methodcomprising: (a) crossing a first plant of lettuce cultivar Cardinal witha second lettuce plant that comprises a desired trait to produce F₁progeny; (b) selecting an F₁ progeny that comprises the desired trait;(c) crossing the selected F₁ progeny with lettuce cultivar Cardinal toproduce backcross progeny; and (d) selecting backcross progenycomprising the desired trait to produce a plant derived from lettucecultivar Cardinal comprising a desired trait. In embodiments, theselected progeny comprises all or essentially all the morphological andphysiological characteristics of the first plant of lettuce cultivarCardinal. Optionally, the method further comprises: (e) repeating steps(c) and (d) one or more times in succession (e.g., one to three, one tofive, one to six, one to seven, one to ten, three to five, three to six,three to seven, three to eight or three to ten times) to produce a plantderived from lettuce cultivar Cardinal comprising the desired trait.

In representative embodiments, the invention also provides a method ofproducing a plant of lettuce cultivar Cardinal comprising a desiredadded trait, the method comprising introducing a transgene conferringthe desired trait into a plant of lettuce cultivar Cardinal. Thetransgene can be introduced by transformation methods (e.g., geneticengineering) or breeding techniques. In embodiments, the plantcomprising the transgene comprises all or essentially all of themorphological and physiological characteristics of lettuce cultivarCardinal.

The invention also provides lettuce plants produced by the methods ofthe invention, wherein the lettuce plant has the desired added trait aswell as seed from such lettuce plants.

According to the foregoing methods, the desired added trait can be anysuitable trait known in the art including, for example, male sterility,male fertility, herbicide resistance, insect or pest (e.g., insectand/or nematode) resistance, modified fatty acid metabolism, modifiedcarbohydrate metabolism, disease resistance (e.g., for bacterial, fungaland/or viral disease), enhanced nutritional quality, increasedsweetness, increased flavor, improved ripening control, improved salttolerance, industrial usage, or any combination thereof.

In representative embodiments, a transgene conferring herbicideresistance confers resistance to glyphosate, sulfonylurea,imidazolinone, dicamba, glufosinate, phenoxy proprionic acid,L-phosphinothricin, cyclohexone, cyclohexanedione, triazine,benzonitrile, or any combination thereof.

In representative embodiments, a transgene conferring pest resistance(e.g., insect and/or nematode resistance) encodes a Bacillusthuringiensis endotoxin.

In representative embodiments, transgenic plants, single locus convertedplants, hybrid plants and lettuce plants derived from lettuce cultivarCardinal have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of themorphological and physiological characteristics of lettuce cultivarCardinal (e.g., as described in Table 1 and Tables 2-12) in anycombination, or even all or essentially all of the morphological andphysiological characteristics of lettuce cultivar Cardinal, so that saidplants are not significantly different for said traits than lettucecultivar Cardinal, as determined at the 5% significance level when grownin the same environmental conditions; optionally, with the presence ofone or more desired additional traits (e.g., male sterility, diseaseresistance, pest or insect resistance, herbicide resistance, and thelike).

In embodiments, the plants of the invention have at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more of the morphological and physiologicalcharacteristics of lettuce cultivar Cardinal (e.g., as described inTable 1 and Tables 2-12).

The invention also encompasses plant parts, plant material, pollen,ovules, leaves, fruit and seed from the lettuce plants of the invention.Also provided is a tissue culture of regenerable cells from the lettuceplants of the invention, where optionally, the regenerable cells are:(a) embryos, meristem, leaves, pollen, cotyledons, hypocotyls, roots,root tips, anthers, flowers, pistils, ovules, seed, shoots, stems,stalks, petioles, pith and/or capsules; or (b) callus or protoplastsderived from the cells of (a). Further provided are lettuce plantsregenerated from a tissue culture of the invention.

In still yet another aspect, the invention provides a method ofdetermining a genetic characteristic of lettuce cultivar Cardinal or aprogeny thereof, e.g., a method of determining a genotype of lettucecultivar Cardinal or a progeny thereof using molecular genetictechniques. In embodiments, the method comprises detecting in the genomeof a Cardinal plant, or a progeny plant thereof, at least a firstpolymorphism, e.g., comprises nucleic acid amplification and/or nucleicacid sequencing. To illustrate, in embodiments, the method comprisesobtaining a sample of nucleic acids from the plant and detecting atleast a first polymorphism in the nucleic acid sample (e.g., using oneor more molecular markers). Optionally, the method may comprisedetecting a plurality of polymorphisms (e.g., two or more, three ormore, four or more, five or more, six or more, eight or more or ten ormore polymorphisms, etc.) in the genome of the plant. In representativeembodiments, the method further comprises storing the results of thestep of detecting the polymorphism(s) on a computer readable medium. Theinvention further provides a computer readable medium produced by such amethod.

In addition to the exemplary aspects and embodiments described above,the invention is described in more detail in the description of theinvention set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of a novellettuce cultivar having desirable characteristics including short corelength, bolting tolerance, and resistances to Bremia lactucae strainsBI: BL: 5-9US, Tomato Bushy Stunt virus, and Nasonovia ribisnigribiotype Nr: 0.

It should be appreciated that the invention can be embodied in differentforms and should not be construed as limited to the embodiments setforth herein. Rather, these embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Unless the context indicates otherwise, it is specifically intended thatthe various features and embodiments of the invention described hereincan be used in any combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a composition comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” when used in a claim or thedescription of this invention is not intended to be interpreted to beequivalent to “comprising.”

“Allele”. An allele is any of one or more alternative forms of a gene,all of which relate to a trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

“Backcrossing”. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

“Big Vein virus”. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

“Bolting”. The premature development of a flowering stalk, andsubsequent seed, before a plant produces a food crop. Bolting istypically caused by late planting when temperatures are low enough tocause vernalization of the plants.

“Bremia lactucae”. An Oomycete that causes downy mildew in lettuce incooler growing regions.

“Core length”. Length of the internal lettuce stem measured from thebase of the cut and trimmed head to the tip of the stem.

“Corky root”. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

“Cotyledon”. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

“Double haploid line”. A stable inbred line achieved by doubling thechromosomes of a haploid line, e.g., from anther culture. For example,some pollen grains (haploid) cultivated under specific conditionsdevelop plantlets containing 1 n chromosomes. The chromosomes in theseplantlets are then induced to “double” (e.g., using chemical means)resulting in cells containing 2n chromosomes. The progeny of theseplantlets are termed “double haploid” and are essentially notsegregating any more (e.g., are stable). The term “double haploid” isused interchangeably herein with “dihaploid.”

“Essentially all of the physiological and morphologicalcharacteristics”. A plant having “essentially all of the physiologicaland morphological characteristics” (and similar phrases) means a planthaving all of the physiological and morphological characteristics oflettuce cultivar Cardinal, except for the characteristic(s) derived froma converted locus/loci (e.g., a single converted locus), for example,introduced via backcrossing to variety Cardinal, a modified gene(s)resulting from genome editing techniques, an introduced transgene (i.e.,introduced via genetic transformation techniques) or mutation, when bothplants are grown under the same environmental conditions. Inembodiments, a plant having “essentially all of the physiological andmorphological characteristics” means a plant having all of thecharacteristics of the reference plant with the exception of five orfewer traits, 4 or fewer traits, 3 or fewer traits, 2 or fewer traits,or one trait. In embodiments, a plant having “essentially all of thephysiological and morphological characteristics” (and similar phrases)optionally has the physiological and morphological characteristicsdescribed in Table 1.

“First water date”. The date the seed first receives adequate moistureto germinate. This can and often does equal the planting date.

“Gene”. As used herein, “gene” refers to a segment of nucleic acidcomprising an open reading frame. A gene can be introduced into a genomeof a species, whether from a different species or from the same species,using transformation or various breeding methods.

“Head diameter”. Diameter of the cut and trimmed head, slicedvertically, and measured at the widest point perpendicular to the stem.

“Head height”. Height of the cut and trimmed head, sliced vertically,and measured from the base of the cut stem to the cap leaf.

“Head weight”. Weight of saleable lettuce head, cut and trimmed tomarket specifications.

“Inbred line”: As used herein, the phrase “inbred line” refers to agenetically homozygous or nearly homozygous population. An inbred line,for example, can be derived through several cycles of sib crossingand/or selfing and/or via double haploid production. In someembodiments, inbred lines breed true for one or more traits of interest.An “inbred plant” or “inbred progeny” is an individual sampled from aninbred line.

“Lettuce Mosaic virus”. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

“Maturity date”. Maturity refers to the stage when the plants are offull size and/or optimum weight and/or in marketable form to be ofcommercial or economic value.

“Nasonovia ribisnigri”. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

“Plant.” As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, fruit, stems, and the like.

“Plant material”. The terms “plant material” and “material obtainablefrom a plant” are used interchangeably herein and refer to any plantmaterial obtainable from a plant including without limitation, leaves,stems, roots, flowers or flower parts, fruits, pollen, ovules, zygotes,seeds, cuttings, cell or tissue cultures, or any other part or productof the plant.

“Plant part”. As used herein, a “plant part” includes any part, organ,tissue or cell of a plant including without limitation an embryo,meristem, head, leaf, pollen, cotyledon, hypocotyl, root, root tip,anther, flower, flower bud, pistil, ovule, seed, shoot, stem, stalk,petiole, pith, capsule, a scion, a rootstock and/or a fruit includingcallus and protoplasts derived from any of the foregoing.

“Quantitative Trait Loci”. Quantitative Trait Loci (QTL) refers togenetic loci that control to some degree, numerically representabletraits that are usually continuously distributed.

“Ratio of head height/diameter”. Head height divided by the headdiameter is an indication of the head shape; <1 is flattened, 1=round,and >1 is pointed.

“Regeneration”. Regeneration refers to the development of a plant fromtissue culture.

“Resistance”. As used herein the terms “resistance” and “tolerance” (andgrammatical variations thereof) are used interchangeably to describeplants that show reduced or essentially no symptoms to a specific biotic(e.g., a pest, pathogen or disease) or abiotic (e.g., exogenous orenvironmental, including herbicides) factor or stressor. In someembodiments, “resistant” or “tolerant” plants show some symptoms but arestill able to produce marketable product with an acceptable yield, e.g.,the yield may still be reduced and/or the plants may be stunted ascompared with the yield or growth in the absence of the biotic and/orabiotic factor or stressor. Those skilled in the art will appreciatethat the degree of resistance or tolerance may be assessed with respectto a plurality or even an entire field of plants. A lettuce plant may beconsidered “resistant” or “tolerant” if resistance/tolerance is observedover a plurality of plants (e.g., an average), even if particularindividual plants may be susceptible to the biotic or abiotic factor orstressor.

“RHS”. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

“Single locus converted”. A single locus converted or conversion plantrefers to a plant that is developed by plant breeding techniques (e.g.,backcrossing), genome editing techniques, genetic transformationtechniques and/or mutation techniques wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single locus introduced into the line viathe plant breeding, genome editing, genetic transformation, or mutationtechniques.

“Substantially equivalent characteristic”. A characteristic that, whencompared, does not show a statistically significant difference (e.g.,p=0.05) from the mean.

“Tip burn”. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

“Tomato Bushy Stunt”. Also called “lettuce necrotic stunt”. A diseasethat causes stunting of growth and leaf mottling.

“Transgene”. A nucleic acid of interest that can be introduced into thegenome of a plant by genetic engineering techniques (e.g.,transformation) or breeding. The transgene can be from the same or adifferent species. If from the same species, the transgene can be anadditional copy of a native coding sequence or can present the nativesequence in a form or context (e.g., different genomic location and/orin operable association with exogenous regulatory elements such as apromoter) than is found in the native state. The transgene can comprisean open reading frame encoding a polypeptide or can encode a functionalnon-translated RNA (e.g., RNAi).

Botanical Description of the Lettuce Cultivar Cardinal (LRLU20-2023).

Characteristics. Romaine lettuce Cardinal is a light green Cos lettucevariety suitable for organic and conventional romaine lettuce productionsettings. Lettuce cultivar Cardinal performs well in Spring and Summerin the coastal areas of California, and in Spring season in the desertSouthwest of California and Arizona. Lettuce cultivar Cardinal is aresult of a cross between a commercial romaine lettuce variety and anearly generation individual plant selected from an elite-by-elite cross.Cardinal was developed with numerous and subsequent generations ofindividual plant selections chosen for horticultural traits such asrefined romaine hearts, short core, color, straight midribs, goodinternal fill, and density. Cardinal possesses strong resistances forall Downy mildew races presently designated in the United States,Nasonovia aphid biotype 0 and Tomato bushy stunt virus.

Lettuce variety Cardinal has shown uniformity and stability for thetraits, within the limits of environmental influence. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The variety has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected in variety Cardinal.

TABLE 1 Select distinguishing characteristics of cultivar CardinalCharacteristic Expression Core length Short Bolting Tolerant, especiallysummer and spring to summer transition Bremia lactucae: Bl: 5-9US Highlyresistant Tomato Bushy Stunt virus Highly resistant Nasonovia ribisnigribiotype Nr: 0 Highly resistant

TABLE 2 Variety Description Information Plant Type: Romaine Seed Seedcolor: Black Light dormancy: Light not required Heat dormancy:Susceptible Cotyledon to Fourth Leaf Stage Shape of cotyledons: BroadUndulation: Flat Anthocyanin distribution: Absent Rolling: AbsentCupping: Uncupped Reflexing: None Mature Leaves Margin incision depth:Moderate Margin indentation: Entire Margin undulation of the apicalmargin: Moderate Green color: Medium green Anthocyanin distribution:Absent Glossiness: Dull Leaf blistering: Absent Trichomes: Absent Leafthickness: Thick Plant at Market Stage Head shape: Elongate Head sizeclass: Large Head weight (g): 797 Head firmness: Moderate Core Diameterat base of head (mm): 39 Core height from base of head to apex (mm): 57Maturity (days) Summer: 70 days Winter: 105 days Adaptation Primary U.S.Regions of Adaptation (tested and proven adapted) Southwest (California,Arizona desert): Yes West Coast: Yes Southeast: Not tested Northeast:Not tested Spring area: Salinas, Santa Maria and San Benito(California); and Yuma (Arizona) Summer area: Salinas (California) Fallarea: Salinas (California) Winter area: Salinas (California) Greenhouse:All year round Soil type: Both of mineral and organic Disease and StressReactions Virus Tomato Bushy Stunt virus (TBSV): Highly resistant Bigvein: Not tested Lettuce Mosaic: Not tested Cucumber Mosaic virus: Nottested Broad Bean Wilt: Not tested Turnip Mosaic virus: Not tested BestWestern Yellows: Not tested Lettuce Infectious Yellows: Not testedFungal/Bacterial Corky Root Rot (Pythium Root Rot): Not tested Bremialactucae (Downy Mildew): Highly resistant: Bl: Bl: 5-9US (resistance toall currently designated races in the United States) Powdery Mildew: Nottested Sclerotinia Rot: Not tested Bacterial Soft Rot (Pseudomonas spp.& others): Not tested Botrytis (Gray Mold): Not tested Insects Nasonoviaribisnigri biotype Nr: 0: Highly resistant Cabbage Loopers: Not testedRoot Aphids: Not tested Green Peach Aphid: Not testedPhysiological/Stress Tip burn: Moderately tolerant Heat: IntermediateDrought: Not tested Cold: Tolerant Salt: Not tested Brown Rib: ResistantBolting: Tolerant, especially in Summer and Spring to Summer transitionPost-Harvest Pink Rib: Not tested Russett Spotting: Not tested RustyBrown Discoloration: Resistant Internal Rib Necrosis (Blackheart, GrayRib, Gray Streak): Resistant Brown Stain: Not tested

TABLE 3 Fourth Leaf Length (mm) at 20 Days LRLU20-2023 Rawhide 61 97 7873 82 88 85 54 101 45 100 50 109 35 88 37 95 77 90 43 110 36 106 49 9464 86 74 74 75 95 52 92 59 84 39 84 21 93 63 AOV Table df SS MS FP-Value Variety 1 11424.4 11424.400 43.053 <0.001 Residuals 38 10083.5265.355 ANOVA shows a significant difference (p < 0.05) in Fourth LeafLength (mm). Duncan Grouping Mean Duncan Grouping LRLU20-2023 90.35 aRawhide 56.55 b Mean std r Min Max Q25 Q50 Q75 LRLU20-2023 90.35 12.0020 61 110 84 91 96.25 Rawhide 56.55 19.66 20 21 97 42 53 73.25

TABLE 4 Fourth Leaf Width (mm) at 20 Days LRLU20-2023 Rawhide 19 21 2017 22 20 25 12 26 11 26 16 26 6 23 15 26 22 24 14 24 10 26 14 20 17 2216 21 17 22 14 22 16 20 11 26 8 23 17 AOV Table df SS MS F P-ValueVariety 1 714.025 714.025 62.125 <0.001 Residuals 38 436.750 11.493ANOVA shows a significant difference (p < 0.05) in Fourth Leaf Width(mm). Duncan Grouping Mean Duncan Grouping LRLU20-2023 23.15 A Rawhide14.70 B Mean std r Min Max Q25 Q50 Q75 LRLU20-2023 23.15 2.41 20 19 2621.75 23.0 26 Rawhide 14.70 4.14 20 6 22 11.75 15.5 17

TABLE 5 Cotyledon Length (mm) at 20 Days LRLU20-2023 Rawhide 14 23 17 1818 18 17 22 14 20 18 17 17 22 18 21 16 25 17 19 16 19 18 19 16 15 18 1917 17 19 18 15 18 14 25 16 23 16 18 AOV Table df SS MS F P-Value Variety1 105.625 105.625 21.796 <0.001 Residuals 38 184.150 4.846 ANOVA shows asignificant difference (p < 0.05) in Cotyledon Length (mm). DuncanGrouping Mean Duncan Grouping Rawhide 19.80 a LRLU20-2023 16.55 b Meanstd r Min Max Q25 Q50 Q75 LRLU20-2023 16.55 1.47 20 14 19 16 17 18Rawhide 19.80 2.75 20 15 25 18 19 22

TABLE 6 Cotyledon Width (mm) at 20 Days LRLU20-2023 Rawhide 6 12 6 11 612 6 13 6 12 7 11 6 12 6 13 5 12 6 13 6 10 7 11 6 10 6 12 7 13 6 11 6 106 11 5 12 6 9 AOV Table df SS MS F P-Value Variety 1 297.025 297.025376.86 <0.001 Residuals 38 29.950 0.788 ANOVA shows a significantdifference (p < 0.05) in Cotyledon Width (mm). Duncan Grouping MeanDuncan Grouping RAWHIDE 11.50 a LRLU20-2023 6.05 b Mean std r Min MaxQ25 Q50 Q75 LRLU20-2023 6.05 0.51 20 5 7 6 6 6 RAWHIDE 11.50 1.15 20 913 11 12 12

TABLE 7 Cotyledon Length to Width Index at 20 Days LRLU20-2023 RAWHIDE2.33 1.92 2.83 1.64 3.00 1.50 2.83 1.69 2.33 1.67 2.57 1.55 2.83 1.833.00 1.62 3.20 2.08 2.83 1.46 2.67 1.90 2.57 1.73 2.67 1.50 3.00 1.582.43 1.31 3.17 1.64 2.50 1.80 2.33 2.27 3.20 1.92 2.67 2.00 AOV Table dfSS MS F P-Value Variety 1 10.353 10.353 150.215 <0.001 Residuals 382.619 0.069 ANOVA shows a significant difference (p < 0.05) in CotyledonL: W Index. Duncan Grouping Mean Duncan Grouping LRLU20-2023 2.75 aRAWHIDE 1.73 b Mean std r Min Max Q25 Q50 Q75 LRLU20-2023 2.75 0.29 202.33 3.20 2.55 2.75 3.0 RAWHIDE 1.73 0.23 20 1.31 2.27 1.57 1.68 1.9

TABLE 8 Head Weight (g) at Fresh Harvest Maturity? Summer, Salinas (CA)Fall, Salinas (CA) LRLU20- Rawhide LRLU20- Rawhide 871.7 640.6 994.81044.2 714.2 1098.0 936.3 1045.9 680.8 726.8 938.1 825.0 558.1 1052.6846.6 872.9 830.9 1044.7 838.6 680.2 981.1 712.5 760.7 1085.2 839.61035.1 731.8 772.2 621.2 921.1 815.2 595.1 911.9 911.2 898.2 916.6 557.4964.1 826.8 705.7 703.3 983.4 694.6 1403.9 809.1 884.7 864.2 946.0 675.41023.8 749.5 801.3 842.5 1015.0 971.0 667.2 737.0 636.9 873.2 858.91030.5 812.7 852.3 1205.7 718.9 871.0 838.0 826.9 662.9 893.7 828.6909.5 618.4 865.8 787.1 1096.7 771.6 844.9 706.1 857.2 AOV Table df SSMS F P-Value Variety 1 217017.36 217017.36 10.581 0.002 Location 140181.13 40181.13 1.959 0.166 Variety: Location 1 25830.08 25830.081.259 0.265 Residuals 76 1558840.47 20511.06 ANOVA shows a significantdifference (p < 0.05) in Head Weight (g). Duncan Grouping Mean DuncanGrouping Rawhide 901.37 a LRLU20-2023 797.20 b Mean std r Min Max Q25Q50 Q75 LRLU20-2023 797.20 116.47 40 557.4 1030.5 712.18 821.0 866.08Rawhide 901.37 167.63 40 595.1 1403.9 809.85 889.2 1026.62

TABLE 9 Plant Height (cm) at Fresh Harvest Maturity Summer, Salinas (CA)Fall, Salinas (CA) LRLU20- Rawhide LRLU20- Rawhide 34 29 38 36 31 34 3835 31 33 38 40 29 33 40 34 34 31 38 36 33 33 37 35 32 33 37 39 31 34 3734 33 33 39 38 29 32 38 32 34 31 39 36 33 32 39 37 31 33 38 38 33 34 4236 32 32 37 38 33 34 35 39 34 31 37 41 34 32 37 39 33 32 39 35 34 31 3934 AOV Table df SS MS F P-Value Variety 1 12.012 12.012 4.040 0.048Location 1 495.013 495.013 166.501 0.000 Variety: Location 1 10.51310.513 3.536 0.064 Residuals 76 225.950 2.973 ANOVA shows a significantdifference (p < 0.05) in plant height (cm). Duncan Grouping Mean DuncanGrouping LRLU20-2023 35.25 a Rawhide 34.48 b Mean std r Min Max Q25 Q50Q75 LRLU20-2023 35.25 3.26 40 29 42 33 34.5 38 Rawhide 34.48 2.86 40 2941 32 34.0 36

TABLE 10 Core Length (mm) at Fresh Harvest Maturity Summer, Salinas (CA)Fall, Salinas (CA) LRLU20- Rawhide LRLU20- Rawhide 50 53 69 76 48 81 5268 47 71 68 55 42 62 70 86 81 62 57 58 69 71 55 66 54 61 55 60 46 76 6148 61 87 78 70 37 71 60 35 49 76 56 87 65 85 54 63 40 85 48 70 52 100 6459 60 87 60 65 65 92 51 61 57 85 58 83 52 56 60 75 58 85 61 82 51 53 6882 AOV Table df SS MS F P-Value Variety 1 3906.013 3906.013 29.239<0.001 Location 1 10.513 10.513 0.079 0.780 Variety: Location 1 918.013918.013 6.872 0.011 Residuals 76 10152.850 133.590 ANOVA shows asignificant difference (p < 0.05) in Core Length (mm). Duncan GroupingMean Duncan Grouping Rawhide 71.20 a LRLU20-2023 57.23 b Mean std r MinMax Q25 Q50 Q75 LRLU20-2023 57.23 9.56 40 37 81 51 57 61.75 Rawhide71.20 13.89 40 35 100 61 71 83.50

TABLE 11 Core Width (mm) at Fresh Harvest Maturity Summer, Salinas (CA)Fall, Salinas (CA) LRLU20- Rawhide LRLU20- Rawhide 43 38 42 40 40 43 4042 37 36 40 40 35 42 38 45 36 45 38 38 43 45 42 46 41 45 40 40 35 42 3840 43 45 42 39 32 45 40 35 38 36 38 41 36 43 37 38 42 44 39 40 37 42 4036 41 42 40 38 45 41 36 45 37 42 42 40 38 52 38 40 32 41 38 42 33 41 3240 AOV Table df SS MS F P-Value Variety 1 154.012 154.012 14.969 <0.001Location 1 10.513 10.513 1.022 0.315 Variety: Location 1 46.512 46.5124.521 0.037 Residuals 76 781.950 10.289 ANOVA shows a significantdifference (p < 0.05) in Core Width (mm). Duncan Grouping Mean DuncanGrouping Rawhide 41.38 a LRLU20-2023 38.60 b Mean std r Min Max Q25 Q50Q75 LRLU20-2023 38.60 3.22 40 32 45 37 38 41.00 Rawhide 41.38 3.34 40 3552 40 41 43.25

TABLE 12 Core Index at Fresh Harvest Maturity Summer, Salinas (CA) Fall,Salinas (CA) LRLU20- LRLU20- 2023 Rawhide 2023 Rawhide 1.16 1.39 1.641.90 1.20 1.88 1.30 1.62 1.27 1.97 1.70 1.38 1.20 1.48 1.84 1.91 2.251.38 1.50 1.53 1.60 1.58 1.31 1.43 1.32 1.36 1.38 1.50 1.31 1.81 1.611.20 1.42 1.93 1.86 1.79 1.16 1.58 1.50 1.00 1.29 2.11 1.47 2.12 1.811.98 1.46 1.66 0.95 1.93 1.23 1.75 1.41 2.38 1.60 1.64 1.46 2.07 1.501.71 1.44 2.24 1.42 1.36 1.54 2.02 1.38 2.08 1.37 1.08 1.58 1.88 1.812.07 1.61 1.95 1.55 1.29 2.13 2.05 AOV Table df SS MS F P-Value Variety1 1.116 1.116 12.838 0.001 Location 1 0.002 0.002 0.027 0.871 Variety:Location 1 0.261 0.261 3.002 0.087 Residuals 76 6.609 0.087 ANOVA showsa significant difference (p < 0.05) in Core Index. Duncan Grouping MeanDuncan Grouping Rawhide 1.72 a LRLU20-2023 1.49 b Mean std r Min Max Q25Q50 Q75 LRLU20-2023 1.49 0.26 40 0.95 2.25 1.31 1.46 1.60 Rawhide 1.720.33 40 1.00 2.38 1.47 1.77 1.97

Tissue Culture.

Further reproduction of lettuce plants variety can occur by tissueculture and regeneration. Tissue culture of various tissues of lettuceand regeneration of plants therefrom is well known and widely published.For example, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants havingdesired characteristics of lettuce cultivar Cardinal. Optionally,lettuce plants can be regenerated from the tissue culture of theinvention comprising all or essentially all of the physiological andmorphological characteristics of lettuce cultivar Cardinal.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques.

Additional Breeding Methods.

This invention is also directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a plant oflettuce cultivar Cardinal. Further, both first and second parent lettucecan come from lettuce cultivar Cardinal. Thus, any of the followingexemplary methods using lettuce cultivar Cardinal are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, double haploid production, and the like. All plantsproduced using lettuce cultivar Cardinal as at least one parent arewithin the scope of this invention, including those developed fromlettuce plants derived from lettuce cultivar Cardinal. Advantageously,lettuce cultivar Cardinal can be used in crosses with other, different,lettuce plants to produce the first generation (F₁) lettuce hybrid seedsand plants with desirable characteristics. The lettuce plants of theinvention can also be used for transformation where exogenous transgenesare introduced and expressed by the plants of the invention. Geneticvariants created either through traditional breeding methods or throughtransformation of the cultivars of the invention by any of a number ofprotocols known to those of skill in the art are intended to be withinthe scope of this invention.

The following describes exemplary breeding methods that may be used withlettuce cultivar Cardinal in the development of further lettuce plants.One such embodiment is a method for developing lettuce cultivar Cardinalprogeny lettuce plants in a lettuce plant breeding program comprising:obtaining a plant, or a part thereof, of lettuce cultivar Cardinal,utilizing said plant or plant part as a source of breeding material, andselecting a lettuce cultivar Cardinal progeny plant with molecularmarkers in common with lettuce cultivar Cardinal and/or with some, allor essentially all of the morphological and/or physiologicalcharacteristics of lettuce cultivar Cardinal (see, e.g., Table 1 andTables 2-12). In representative embodiments, the progeny plant has atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the morphological andphysiological characteristics of lettuce cultivar Cardinal (e.g., asdescribed in Table 1 and Tables 2-12) or even all of the morphologicaland physiological characteristics of lettuce cultivar Cardinal so thatsaid progeny lettuce plant is not significantly different for saidtraits than lettuce cultivar Cardinal, as determined at the 5%significance level when grown in the same environmental conditions;optionally, with the presence of one or more desired additional traits(e.g., male sterility, disease resistance, pest or insect resistance,herbicide resistance, and the like). Breeding steps that may be used inthe breeding program include pedigree breeding, backcrossing, mutationbreeding and/or recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers) and/or and the making of doublehaploids may be utilized.

Another representative method involves producing a population of lettucecultivar Cardinal progeny plants, comprising crossing lettuce cultivarCardinal with another lettuce plant, thereby producing a population oflettuce plants that, on average, derives 50% of its alleles (i.e., TAC)from lettuce cultivar Cardinal. A plant of this population may beselected and repeatedly selfed or sibbed with a lettuce plant resultingfrom these successive filial generations or backcrossed to lettucecultivar Cardinal. Another approach is to make double haploid plants toachieve homozygosity. One embodiment of this invention is a lettuceplant produced by these methods and that has obtained at least 50% ofits alleles from lettuce cultivar Cardinal. In embodiments, the methodsof the invention produce a population of lettuce plants that, onaverage, derives at least 6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of itsalleles (i.e., TAC) from lettuce cultivar Cardinal, e.g., at least about6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the genetic complement of lettucecultivar Cardinal. One representative embodiment of this invention isthe lettuce plant produced by the methods of the invention and that hasobtained at least 6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of its alleles (i.e.,TAC) from lettuce cultivar Cardinal, and optionally is the result of abreeding process comprising one or two breeding crosses and one or moreof selfing, sibbing, backcrossing and/or double haploid techniques inany combination and any order. In embodiments, the breeding process doesnot include a breeding cross, and comprises selfing, sibbing,backcrossing and or double haploid technology.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). In embodiments, the inventionencompasses Cardinal progeny plants having a combination of at least 2,3, 4, 5, 6, 7, 8, 9, 10 or more of the characteristics as describedherein for lettuce cultivar Cardinal, so that said progeny lettuce plantis not significantly different for said traits than lettuce cultivarCardinal, as determined at the 5% significance level when grown in thesame environmental conditions. Using techniques described herein andthose known in the art, molecular markers may be used to identify saidprogeny plant as progeny of lettuce cultivar Cardinal. Mean trait valuesmay be used to determine whether trait differences are significant, andoptionally the traits are measured on plants grown under the sameenvironmental conditions.

Progeny of lettuce cultivar Cardinal may also be characterized throughtheir filial relationship with lettuce cultivar Cardinal, as forexample, being within a certain number of breeding crosses of lettucecultivar Cardinal. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross or a backcross to Cardinal as a recurrent parent,made to select among existing genetic alleles. The lower the number ofbreeding crosses in the pedigree, the closer the relationship betweenlettuce cultivar Cardinal and its progeny. For example, progeny producedby the methods described herein may be within 1, 2, 3, 4, 5 or morebreeding crosses of lettuce cultivar Cardinal.

In representative embodiments, a lettuce plant derived from lettucecultivar Cardinal comprises cells comprising at least one set ofchromosomes derived from lettuce cultivar Cardinal. In embodiments, thelettuce plant or population of lettuce plants derived from lettucecultivar Cardinal comprises, on average, at least 6.25%, 12.5%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% of its alleles (i.e., TAC) from lettuce cultivarCardinal, e.g., at least about 6.25%, 12.5%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of thegenetic complement of lettuce cultivar Cardinal, and optionally is theresult of a breeding process comprising one or two breeding crosses andone or more of selfing, sibbing, backcrossing and/or double haploidtechniques in any combination and any order. In embodiments, thebreeding process does not include a breeding cross, and comprisesselfing, sibbing, backcrossing and or double haploid technology. Inembodiments, the lettuce plant derived from lettuce cultivar Cardinal isone, two, three, four, five or more breeding crosses removed fromlettuce cultivar Cardinal.

In representative embodiments, a plant derived from lettuce cultivarCardinal is a double haploid plant, a hybrid plant or an inbred plant.

In embodiments, a hybrid or derived plant from lettuce cultivar Cardinalcomprises a desired added trait. In representative embodiments, alettuce plant derived from lettuce cultivar Cardinal comprises all ofthe morphological and physiological characteristics of lettuce cultivarCardinal (e.g., as described in Table 1 and Tables 2-12). Inembodiments, the lettuce plant derived from lettuce cultivar Cardinalcomprises essentially all of the morphological and physiologicalcharacteristics of lettuce cultivar Cardinal (e.g., as described inTable 1 and Tables 2-12) in any combination, with the addition of adesired added trait.

Those skilled in the art will appreciate that any of the traitsdescribed above with respect to plant transformation methods can beintroduced into a plant of the invention (e.g., lettuce cultivarCardinal and hybrid lettuce plants and other lettuce plants derivedtherefrom) using breeding techniques.

Genetic Transformation.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign nucleic acids including additional or modified versions ofnative (endogenous) nucleic acids (optionally driven by a non-nativepromoter) in order to alter the traits of a plant in a specific manner.Any nucleic acid sequences, whether from a different species, the samespecies or an artificial sequence, which are introduced into the genomeusing transformation or various breeding methods, are referred to hereincollectively as “transgenes.” Over the last fifteen to twenty years,several methods for producing transgenic plants have been developed, andin particular embodiments the present invention also relates totransformed versions of the plants disclosed herein.

Genetic engineering techniques can be used (alone or in combination withbreeding methods) to introduce one or more desired added traits intoplant, for example, lettuce cultivar Cardinal or progeny or lettuceplants derived thereof. Once a transgene has been introduction into aplant by genetic transformation, it can be transferred to other plantsvia conventional breeding.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Optionally, such avector comprises one or more nucleic acids comprising a coding sequencefor a polypeptide or an untranslated functional RNA under control of, oroperatively linked to, a regulatory element (for example, a promoter).In representative embodiments, the vector(s) may be in the form of aplasmid, and can be used alone or in combination with other plasmids, toprovide transformed lettuce plants using transformation methods asdescribed herein to incorporate transgenes into the genetic material ofthe lettuce plant.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct nucleic acid transfermethod, such as microprojectile-mediated delivery (e.g., with abiolistic device), DNA injection, Agrobacterium-mediated transformation,electroporation, and the like. Transformed plants obtained from theplants (and parts and tissue culture thereof) of the invention areintended to be within the scope of this invention.

Expression Vectors for Plant Transformation—Selectable markers.

Expression vectors typically include at least one nucleic acidcomprising or encoding a selectable marker, operably linked to aregulatory element (for example, a promoter) that allows transformedcells containing the marker to be either recovered by negativeselection, e.g., inhibiting growth of cells that do not contain theselectable marker, or by positive selection, e.g., screening for theproduct encoded by the selectable marker. Many commonly used selectablemarkers for plant transformation are well known in the transformationart, and include, for example, nucleic acids that code for enzymes thatmetabolically detoxify a selective chemical agent which may be anantibiotic or an herbicide, or nucleic acids that encode an alteredtarget which is insensitive to the inhibitor. Positive selection methodsare also known in the art.

Commonly used selectable markers in plants include, but are not limitedto: neomycin phosphotransferase II (nptII) conferring resistance tokanamycin, hygromycin phosphotransferase conferring resistance to theantibiotic hygromycin, bacterial selectable markers that conferresistance to antibiotics (e.g., gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, selectable markers conferring resistance to herbicides(e.g., glyphosate, glufosinate, or bromoxynil). Selection of transformedplant cells can also be based on screening presumptively transformedplant cells rather than direct genetic selection of transformed cellsfor resistance to a toxic substance such as an antibiotic; such markersinclude without limitation alpha-glucuronidase (GUS),alpha-galactosidase, luciferase, and Green Fluorescent Protein (GFP) andmutant GFPs.

Expression Vectors for Plant Transformation—Promoters.

Transgenes included in expression vectors are generally driven by anucleotide sequence comprising a regulatory element (for example, apromoter). Numerous types of promoters are well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells.

Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred.” Promoters thatinitiate transcription only in certain tissue are referred to as“tissue-specific.” A “cell type” specific promoter preferentially drivesexpression in certain cell types in one or more organs, for example,vascular cells in roots or leaves. An “inducible” promoter is a promoterthat is under environmental control. Examples of environmentalconditions that may affect transcription by inducible promoters includeanaerobic conditions or the presence of light. Tissue-specific,tissue-preferred, cell type specific, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

Many suitable promoters are known in the art and can be selected andused to achieve the desired outcome.

Signal Sequences for Targeting Proteins to Subcellular Compartments.

Transport of polypeptides produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isgenerally accomplished by means of operably linking a nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of anucleic acid encoding the polypeptide of interest. Signal sequences atthe 5′ and/or 3′ end of the coding sequence target the polypeptide toparticular subcellular compartments.

The presence of a signal sequence can direct a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J, 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Polypeptide Transgenes and Agronomic Transgenes.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign polypeptide then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981). According to a representative embodiment, the transgenic plantprovided for commercial production of foreign protein is a lettuce plantof the invention. In another embodiment, the biomass of interest is seedand/or fruit.

Likewise, by means of the present invention, agronomic transgenes andother desired added traits can be expressed in transformed plants (andtheir progeny, e.g., produced by breeding methods). More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest or other desired added traits. Exemplary nucleicacids of interest in this regard conferring a desired added trait(s)include, but are not limited to, those transgenes that confer resistanceto confer resistance to plant pests (e.g., nematode or insect) ordisease (e.g., fungal, bacterial or viral), transgenes that conferherbicide tolerance, transgenes that confer male sterility, andtransgenes that confer or contribute to a value-added trait such asincreased nutrient content (e.g., iron, nitrate), increased sweetness(e.g., by introducing a transgene coding for monellin), modified fattyacid metabolism (for example, by introducing into a plant an antisensesequence directed against stearyl-ACP desaturase to increase stearicacid content of the plant), modified carbohydrate composition (e.g., byintroducing into plants a transgene coding for an enzyme that alters thebranching pattern of starch), modified fruit color (e.g., external fruitcolor and/or fruit flesh), or modified flavor profile of the fruit.

In embodiments, the transgene encodes a non-translated RNA (e.g., RNAi)that is expressed to produce targeted inhibition of gene expression,thereby conferring the desired trait on the plant.

In embodiments, the transgene encodes the machinery used for geneediting techniques.

Any transgene, including those exemplified above, can be introduced intothe lettuce plants of the invention through a variety of meansincluding, but not limited to, transformation (e.g., genetic engineeringtechniques), conventional breeding, and introgression methods tointroduce the transgene into other genetic backgrounds.

Methods for Plant Transformation.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993). Commonly usedplant transformation methods include agrobacterium-mediatedtransformation and direct transgene transfer methods (e.g.,microprojectile-mediated transformation, sonication, liposome orspheroplast fusion, and electroporation of protoplasts or whole cells).

Following transformation of plant target tissues, expression ofselectable marker transgenes (e.g., as described above) allows forpreferential selection of transformed cells, tissues and/or plants,using regeneration and selection methods now well known in the art.

The foregoing methods for transformation are typically used to produce atransgenic lettuce line. The transgenic lettuce line can then be crossedwith another (non-transgenic or transgenic) line in order to produce anew transgenic lettuce line. Alternatively, a transgene that has beenengineered into a particular plant using transformation techniques canbe introduced into another plant or line using traditional breeding(e.g., backcrossing) techniques that are well known in the plantbreeding arts. For example, a backcrossing approach can be used to movean engineered transgene from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign transgenein its genome into an inbred line or lines which do not contain thattransgene. As used herein, “crossing” can refer to a simple X by Ycross, or the process of backcrossing, depending on the context.

Locus Conversions.

When the term “lettuce plant” is used in the context of the presentinvention, this term also includes any locus conversions of that plantor variety. The term “locus converted plant” as used herein refers tothose plants that are developed, for example, by backcrossing, genomeediting, genetic transformation and/or mutation, wherein essentially allof the desired morphological and physiological characteristics of avariety are recovered in addition to the one or more loci introducedinto the variety. To illustrate, backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, e.g.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to the recurrentparent. The parental plant that contributes the locus/loci for thedesired characteristic(s) is termed the “nonrecurrent” or “donorparent.” This terminology refers to the fact that the nonrecurrentparent is generally used one time in the breeding e.g., backcross)protocol and therefore does not recur. The locus that is transferred canbe a native gene, a mutated native gene (e.g., naturally occurring, bychemical or radiation mutagenesis, or by genome editing) or a transgeneintroduced by genetic engineering techniques into the plant (or ancestorthereof). The parental plant into which the locus/loci from thenonrecurrent parent are transferred is known as the “recurrent” parentas it is used for multiple rounds in the backcrossing protocol. Poehlman& Sleper (1994) and Fehr (1993). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the locus/loci of interest tobe transferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a plantis obtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant in addition to the transferred locus/loci andassociated trait(s) from the nonrecurrent parent.

Genetic Analysis of Lettuce Cultivar Cardinal.

The invention further provides a method of determining a geneticcharacteristic of lettuce cultivar Cardinal or a progeny thereof, e.g.,a method of determining a genotype of lettuce cultivar Cardinal or aprogeny thereof. In embodiments, the method comprises detecting in thegenome of a Cardinal plant, or a progeny plant thereof, at least a firstpolymorphism (e.g., using nucleic acid amplification, nucleic acidsequencing and/or one or more molecular markers). To illustrate, inembodiments, the method comprises obtaining a sample of nucleic acidsfrom the plant and detecting at least a first polymorphism in thenucleic acid sample. Optionally, the method may comprise detecting aplurality of polymorphisms (e.g., two or more, three or more, four ormore, five or more, six or more, eight or more or ten or morepolymorphisms, etc.) in the genome of the plant. In representativeembodiments, the method further comprises storing the results of thestep of detecting the polymorphism(s) on a computer readable medium. Theinvention further provides a computer readable medium produced by such amethod.

DEPOSIT INFORMATION

Applicants have made a deposit of at least 625 seeds of lettuce cultivarCardinal with the Provasoli-Guillard National Center for Marine Algaeand Microbiota (NCMA) at Bigelow Laboratory for Ocean Sciences, 60Bigelow Drive, East Boothbay, Me., 04544 U.S.A. under NCMA Accession No.______ on ______. This deposit of lettuce variety Cardinal will bemaintained in the NCMA depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theeffective life of the patent, whichever is longer, and will be replacedif any of the deposited seed becomes nonviable during that period.Additionally, Applicants have satisfied all the requirements of 37C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the samples. Access to this deposit will be made availableduring the pendency of this application to the Commissioner uponrequest. Upon the issuance of a patent on the variety, the variety willbe irrevocably and without restriction released to the public byproviding access to the deposit of at least 2500 seeds of the varietywith the NCMA. Applicants impose no restrictions on the availability ofthe deposited material from the NCMA; however, Applicants have noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicants do notwaive any infringement of its rights granted under this patent or underthe Plant Variety Protection Act (7 USC § 2321 et seq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be apparent that certain changes and modifications suchas single locus modifications and mutations, somaclonal variants,variant individuals selected from large populations of the plants of theinstant inbred and the like may be practiced within the scope of theinvention.

What is claimed is:
 1. A seed of lettuce cultivar Cardinal, arepresentative sample of seed having been deposited under NCMA AccessionNo. ______.
 2. A plant of lettuce cultivar Cardinal, a representativesample of seed having been deposited under NCMA Accession No. ______. 3.A lettuce plant, or a part thereof, having all of the physiological andmorphological characteristics of the lettuce plant of claim
 2. 4. Aprogeny lettuce plant of the plant of claim 2 that comprises at least50% of the alleles of the plant of claim 2, wherein the progeny lettuceplant is characterized by a short core length and high resistances toBremia lactucae strains BI: 5-9US, Tomato Bushy Stunt virus, andNasonovia ribisnigri biotype Nr:
 0. 5. A seed that produces the plant ofclaim
 4. 6. A plant part of the lettuce plant of claim 2, wherein theplant part is a head, leaf, pollen, ovule, anther, root, or cell.
 7. Atissue culture of regenerable cells of the plant of claim
 2. 8. Alettuce plant regenerated from the tissue culture of claim 7, whereinsaid lettuce plant expresses all of the physiological and morphologicalcharacteristics of lettuce cultivar Cardinal.
 9. A converted lettuceplant produced by introducing a single locus conversion into the plantof claim 2, wherein said converted lettuce plant comprises said singlelocus conversion and otherwise comprises all of the physiological andmorphological characteristics of lettuce cultivar Cardinal.
 10. Aprocessed product from the plant of claim 2, wherein the processedproduct comprises cut, sliced, ground, pureed, dried, canned, jarred,washed, packaged, frozen and/or heated leaves.
 11. A method of producinglettuce seed, the method comprising crossing the plant of claim 2 withitself or a second lettuce plant and harvesting the resulting seed. 12.An F1 lettuce seed produced by the method of claim
 12. 13. A F1 lettuceplant, produced by growing the seed of claim
 13. 14. A plant part of theF1 lettuce plant of claim 13, wherein the plant part is a head, leaf,pollen, ovule, anther, root, or cell.
 15. A doubled haploid plantproduced from the F1 lettuce plant of claim
 13. 16. A method forproducing a seed of a lettuce plant derived from the plant of claim 2,the method comprising: (a) crossing a plant of lettuce cultivar Cardinalwith a second lettuce plant; and (b) allowing seed to form; (c) growinga plant from the seed of step (b) to produce a plant derived fromlettuce cultivar Cardinal; (d) selfing the plant of step (c) or crossingit to a second lettuce plant to form additional lettuce seed derivedfrom lettuce cultivar Cardinal; and (e) optionally repeating steps (c)and (d) one or more times to generate further derived lettuce seed fromlettuce cultivar Cardinal, wherein in step (c) a plant is grown from theadditional lettuce seed of step (d) in place of growing a plant from theseed of step (b).
 17. A seed produced by the method of claim 16, whereinthe seed comprises at least 50% of the alleles of lettuce cultivarCardinal and is within one breeding cross of lettuce cultivar Cardinal,and wherein the seed produces a lettuce plant that is characterized by ashort core length and high resistances to Bremia lactucae strains BI:5-9US, Tomato Bushy Stunt virus, and Nasonovia ribisnigri biotype Nr: 0.18. A plant, or part thereof, produced by growing the seed of claim 16.19. A method of vegetatively propagating the plant of claim 2, themethod comprising: (a) collecting tissue capable of being propagatedfrom a plant of lettuce cultivar Cardinal; (b) cultivating the tissue toobtain proliferated shoots; (c) rooting the proliferated shoots toobtain rooted plantlets; and (d) optionally, growing plants from therooted plantlets.
 20. A lettuce plantlet or plant obtained by the methodof claim 19, wherein the lettuce plantlet or plant expresses all of thephysiological and morphological characteristics of lettuce cultivarCardinal.
 21. A method of introducing a desired added trait into lettucecultivar Cardinal, the method comprising: (a) crossing the plant ofclaim 2 with a lettuce plant that comprises a desired added trait toproduce F1 progeny; (b) selecting an F1 progeny that comprises thedesired added trait; (c) crossing the selected F1 progeny with lettucecultivar Cardinal to produce backcross progeny; (d) selecting abackcross progeny comprising the desired added trait; and (e) optionallyrepeating steps (c) and (d) one or more times to produce a plant derivedfrom lettuce cultivar Cardinal comprising a desired added trait andotherwise all of the physiological and morphological characteristics oflettuce cultivar Cardinal, wherein in step (c) the selected backcrossprogeny produced in step (d) is used in place of the selected F1 progenyof step (b).
 22. The method of claim 21, wherein the desired added traitis male sterility, pest resistance, insect resistance, diseaseresistance, herbicide resistance, or any combination thereof.
 23. Alettuce plant produced by the method of claim 21, wherein the lettuceplant has the desired added trait and otherwise all of the physiologicaland morphological characteristics of lettuce cultivar Cardinal.
 24. Aseed of the plant of claim 23, wherein the seed produces a plant thathas the desired added trait and otherwise all of the physiological andmorphological characteristics of lettuce cultivar Cardinal.
 25. A seedthat produces the plant of claim
 23. 26. A method of producing a plantof lettuce cultivar Cardinal comprising a desired added trait, themethod comprising introducing a transgene conferring the desired addedtrait into the plant of claim 2, wherein the plant comprises the desiredadded trait and otherwise all of the physiological and morphologicalcharacteristics of lettuce cultivar Cardinal.
 27. A lettuce plantproduced by the method of claim 26, wherein the lettuce plant comprisesthe desired added trait and otherwise all of the physiological andmorphological characteristics of lettuce cultivar Cardinal.
 28. A seedof the plant of claim 27, wherein the seed produces a plant that has thedesired added trait and otherwise all of the physiological andmorphological characteristics of lettuce cultivar Cardinal.
 29. A methodof determining a genotype of lettuce cultivar Cardinal, the methodcomprising: (a) obtaining a sample of nucleic acids from the plant ofclaim 2; and (b) detecting a polymorphism in the nucleic acid sample.30. A method of producing a lettuce leaf, the method comprising: (a)growing the lettuce plant according to claim 2 to produce a lettuceleaf; and (b) harvesting the lettuce leaf.
 31. A method of producing alettuce leaf, the method comprising: (a) growing the lettuce plantaccording to claim 23 to produce a lettuce leaf; and (b) harvesting thelettuce leaf.
 32. A method of developing a lettuce line in a lettuceplant breeding program using plant breeding techniques, which includeemploying a lettuce plant, or its parts, as a source of plant breedingmaterial, comprising: (a) obtaining the lettuce plant, or its parts, ofclaim 2 as a source of breeding material; and (b) applying plantbreeding techniques.